CRF is the major neuropeptide hormone associated with stress and is mediated predominantly signaling through CRF receptor subtype 1 (CRFR1) in the CNS. CRF signaling has been implicated in Alzheimer's disease (AD) as there is increased CRF expression, CRF binding to and upregulation of CRFR1 receptors in the cortex, hippocampus and hypothalamus appears to occur early in disease AD progression and are prominent neurochemical changes in areas vulnerable to AD neuropathology. CRFR1 expression also intersects with Abeta (A?) and tau pathologies, both known to be critical hallmarks of AD. There have been substantial efforts in academia and pharmaceutical companies to develop potent small molecule antagonists of CRFR1, however, even the current 3rd generation antagonists are still directed against the C- terminal regions of the CRFR1 receptor are still based on the 2nd generation chemical scaffolds along with their inherent toxicity liabilities. This project capitalizes on the molecular model of CRFR1 activation, established through extensive studies, where the first step is binding of CRF to specific ligand binding motifs in the receptor's N-terminal region and binding motifs. We have developed and validated an novel homogeneous TR-FRET assay to measure the direct binding of biotin-labeled CRF to the N-terminal extracellular domain (ECD) of maltose binding protein labeled CRFR1 compatible with very high-throughput screening. We have developed this assay, verified that unlabeled CRF displaces biotin-CRF, and that several known C-terminal CRFR1 antagonists do not displace CRF from the N-term CRFR1 ECD. Motivated by these recent experiments, we propose to screen a large library of compounds against N-terminal CRFR1 ECD in collaboration with the Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys Medical Discovery Institute. In Aim 1, we will fully implement this primary N- terminal CRFR1 ECD assay and complete an HTS campaign to identify and confirm candidate N-terminal CRFR1 antagonists. Aim 2 will validate these hits for potency, specificity, functional antagonism and elucidate emergent structure activity relationships (SAR) to assess their chemical tractability. Aim 3 will advance validated hits through hit-to-lead through cycles of medicinal chemistry and testing through 2 critical path assays for potency and functional antagonism, then additional lead optimization cycles where additionally, potency and efficacy in biologically relevant 3 assays for target engagement (e.g. brain tissue slices). The best 2-3 probe compound(s), will be scale-up to benchmark their ADME/T, PK/PD and brain penetrance and in vivo proof-of-concept studies in mouse AD models.